NG-Aminoguanidines from Primary Amines and the Preparation of

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J. Med. Chem. 2008, 51, 924–931

NG-Aminoguanidines from Primary Amines and the Preparation of Nitric Oxide Synthase Inhibitors Nathaniel I. Martin,† William T. Beeson, Joshua J. Woodward, and Michael A. Marletta* Departments of Chemistry, Molecular and Cellular Biology, and DiVision of Physical Sciences, Lawrence Berkeley National Laboratory, UniVersity of California, Berkeley, Berkeley, California 94720-3220 ReceiVed September 9, 2007

A concise, general, and high-yielding method for the preparation of NG-aminoguanidines from primary amines is reported. Using available and readily prepared materials, primary amines are converted to protected NG-aminoguanidines in a one-pot procedure. The method has been successfully applied to a number of examples including the syntheses of four nitric oxide synthase (NOS) inhibitors. The inhibitors prepared were investigated as competitive inhibitors and as mechanistic inactivators of the inducible isoform of NOS (iNOS). In addition, one of the four inhibitors prepared, NG-amino-NG-2,2,2-trifluoroethyl-L-arginine 19, displays the unique ability to both inhibit NO formation and prevent NADPH consumption by iNOS without irreversible inactivation of the enzyme. Scheme 1. Reaction Catalyzed by NOS

Introduction G

N -Aminoguanidines have been of medicinal interest for some time and have been previously described as anticancer, antibacterial, and antiviral agents.1,2 This class of compounds also display dopamine β-oxidase inhibition and antihypertensive properties.3 Substituted aminoguanidines have been shown to inhibit the advanced nonenzymatic glycosylation of proteins4,5 and arylaminoguanidines have been described as a novel class of 5-HT2aA receptor antagonists.6 Recent work has also suggested that NG-aminoguanidine functionalities can be utilized in the design of potent inhibitors for trypsin-like serine proteases.7,8 Our interest in these compounds stems from our continuing work with the nitric oxide synthases (NOSsa , EC 1.14.13.39) and previous reports that certain NG-aminoguanidines inhibit NOS activity.9,10 The NOSs are a family of enzymes that catalyze the conversion of L-arginine to G L-citrulline and nitric oxide (NO) via the intermediate N 11,12 NOSs are active as hydroxy-L-arginine (NHA; Scheme 1). homodimers, with each subunit containing a C-terminal reductase domain (with binding sites for NADPH, FAD, and FMN) and an N-terminal oxygenase domain containing the heme prosthetic group. The substrate, L-arginine, and a redox cofactor, (6R)-5,6,7,8 tetrahydro-L-biopterin (H4B), both bind near the heme center in the oxygenase domain.13 NO regulates numerous physiological processes, including neurotransmission, smooth muscle relaxation, platelet reactivity, and the cytotoxic activity of immune cells.14 Overproduction of NO has been linked to the pathogenesis of a number of disease states, including septic shock, neurodegenerative dis* To whom correspondence should be addressed. Departments of Chemistry and Molecular and Cellular Biology, and Division of Physical Sciences, Lawrence Berkeley National Laboratory, University of California, Berkeley, 570 Stanley Hall, Berkeley, CA 94720-3220. Tel.: (510) 6662763. Fax: (510) 666-2765. E-mail: [email protected]. † Current address: Department of Medicinal Chemistry and Chemical Biology, Utrecht University, F.A.F.C. Wentgebouw, Room Z615, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands. a Abbreviations: NOS, nitric oxide synthase; iNOS, inducible NOS; iNOSheme, heme domain of inducible NOS; H4B, (6R)-5,6,7,8-tetrahydroL-biopterin; NADPH, nicotinamide adenine dinucleotide phosphate; FAD, flavin adenine dinucleotide; FMN, flavin monomucleotide; CbZNCS, N-benzyloxycarbonyl isothiocyanate; OxyMb, oxymyoglobin; DTT, dithiothreitol; HEPES, 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid.

orders, and inflammation.15 For this reason, compounds capable of inhibiting the NOSs, including NG-aminoguanidines, remain of interest. Despite the widespread interest in NG-aminoguanidines, few general methods have been developed by which these functionalities can be conveniently and safely constructed under mild conditions. Traditional approaches involve the use of toxic reagents and harsh reaction conditions and have generally relied upon the addition of hydrazines to cyanimides (prepared by CNBr treatment of the starting amine)16,17 or to thioureas requiring preactivation with HgCl2, PbO, or alkyl iodides.18,19 Recently, Katritzky and co-workers described an alternate route to NG-aminoguanidines via treatment of di(benzotriazol-1yl)methanimine with the appropriate amines and hydrazines in refluxing toluene.20 As mentioned above, NG-aminoguanidine containing compounds have also been described as inhibitors of NOS.9 NGAmino-L-arginine is a well-known irreversible inhibitor of NOS and exhibits mechanism-based, time-dependent inactivation of the enzyme.21 The nature of this inactivation has been previously investigated and has been shown to be the result of a covalent modification of the enzyme. Based on mass spectrometric evidence, Osawa and co-workers have proposed a modification pathway whereby the inhibitor is converted to a reactive intermediate by the action of NOS itself.22 This reactive species then covalently cross-links with the heme prosthetic group, resulting in a catalytically inactive form of the enzyme (Scheme 2). Whether this heme-modification pathway is a general mechanism by which NG-aminoguanidines inhibit NOS is

10.1021/jm701119v CCC: $40.75  2008 American Chemical Society Published on Web 01/26/2008

NG-Aminoguanidines from Primary Amines

Scheme 2. Proposed Inactivation of NOS by NG-Amino-L-arginine22

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 925 Table 1. Cbz-Protected NG-Aminoguanidines Prepared

unknown and presented an additional motivation for our investigation. Results and Discussion An initial objective in our investigation was the development of a convenient and general approach for the synthesis of NGaminoguanidines. While the addition of nucleophiles to thioureas has been previously described in this regard,18,19 the lack of suitable protecting groups along with the use of toxic reagents and harsh coupling conditions has somewhat limited the generality of these approaches. We recently described the preparation and use of N-benzyloxycarbonyl isothiocyanate (CbzNCS) in the conversion of amines to protected NGhydroxyguanidines via thiourea intermediates. The thiourea intermediates need not be isolated and are readily converted to protected NG-hydroxyguanidines in a high-yielding, one-pot procedure.23 We have found that a similar approach, whereby Cbz-protected thioureas are activated with N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDCI) and treated with benzyl carbazate, is of equal generality in the synthesis of NG-aminoguanidines (Scheme 3). Scheme 3. One-Pot Preparation of NG-Aminoguanidines

The strategy illustrated in Scheme 3 was successfully applied to a variety of amines. The expected Cbz-protected NGaminoguanidine products were all rapidly formed at room temperature and in good yields (Table 1). A practical advantage of this approach is that the NG-hydroxyguanidine moiety is installed in a fully Cbz-protected form, allowing for purification by standard chromatographic techniques prior to deprotection. While previously reported syntheses of NG-aminoguanidines have provided the target molecules directly, the lack of protecting groups can complicate purification owing to the high polarity of such compounds. In addition, NG-aminoguanidines are stable to reductive conditions, allowing for global removal of the Cbz groups by standard hydrogenation to directly yield the fully deprotected products (see below for further examples and description of deprotection conditions). It should be noted, however, that it is not possible to convert secondary amines to the corresponding NG-aminoguanidines using this procedure. The EDCI-mediated coupling likely occurs via the formation of a transient carbodiimide24 not attainable with secondary amines. In cases where NG-aminoguanidines derived from

a Isolated yield. b An equivalent of NEt3 added to the amine hydrochloride prior to treatment with CbzNCS.

secondary amines are required, the approach of Katritzy and co-workers presents a viable alternative.20 After establishing the validity of this approach for the preparation of the NG-aminoguanidine core, the methodology was further applied to the preparation of four L-arginine analogues to be investigated as inhibitors of iNOS. The four L-arginine analogues were designed to probe the role of both electron-rich and electron-poor substituents located at either of the two amino positions originally deriving from the hydrazine used in constructing the NG-aminoguanidine center. Benzyl carbazate as well as the commercially available 2,2,2-trifluoroethyl- and monomethyl-substituted hydrazines were used as starting materials to yield the four analogues each deriving from the same thiourea precursor 8. The orthogonally protected L-ornithine species 7 was first prepared by standard means and converted to 8 by TFA deprotection, followed by treatment with CbzNCS (Scheme 4). Scheme 4. Preparation of Thiourea 8

The required hydrazines were then Cbz-protected using standard carbamate chemistry (Scheme 5). In choosing the position for the Cbz protecting group on the hydrazine, it is possible to dictate the substitution pattern in the final NGaminoguanidine product. Compound 9 was prepared by direct treatment of methylhydrazine with CbzCl leading to carbamate

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Scheme 5. Preparation of Cbz-Protected Methyl and 2,2,2-Trifluoroethylhydrazines

protection at the more nucleophilic (methylated) nitrogen. When methylhydrazine was first treated with Boc2O followed by CbzCl, the Cbz group was installed at the less nucleophilic nitrogen. Acid treatment to remove the Boc carbamate then yielded compound 10. Compound 11 was obtained by treating 2,2,2-trifluoroethylhydrazine with CbzCl. In this case, the electron withdrawing effect of the trifluoromethyl group led to preferential protection of the less-substituted nitrogen. The four Cbz-protected NG-amino-L-arginine analogues 12-15 were then prepared by treatment of thiourea 8 with EDCI, NEt3, and the appropriate Cbz-protected hydrazine (Scheme 6). Deprotection of 12-15 was accomplished by hydrogenation over Pd/C to provide the amino acid products 16-19 as the acetate salts after lyophilization (with the exception of compound 18, which was isolated as the free base after lyophilization. as evidenced by NMR; Scheme 6). Scheme 6. Preparation of Amino Acid Products 16–19

Martin et al.

substrate(s) in identical fashion to the full-length enzyme and is catalytically competent when provided with an exogenous source of electrons such as sodium dithionite.25 Addition of 16-19 to H4B-bound iNOSheme induced a low- to high-spin state conversion at the heme center resulting from loss of water (FeIIIaqua f FeIII-unligated) upon binding. This transition leads to a decrease in absorbance at ∼418 nm and an increase in absorbance at ∼394 nm in the UV–visible spectrum. The magnitude of spectral change, ∆∆Abs (where ∆∆Abs ) ∆Abs394 - ∆Abs418), is dependent on the concentration of the added analogue. Spectral Kd values for each compound are then determined by fitting the titration data to the saturation binding equation: ∆∆Abs ) (∆∆Absmax × [compound])/(Kd + [compound]). Table 2 summarizes the spectral binding assay results for compounds 16-19 (graphic depictions of the titration experiments for all four analogs provided in Supporting Information). Table 2. Spectral Kd Values for 16–19 compound

spectral Kda (µM)

L-arginine NG-amino-L-arginine (16) NG-methylamino-L-arginine (17) NG-amino-NG-methyl-L-arginine (18) NG-amino-NG-2,2,2-trifluoroethyl-L-arginine (19)

7.0 ( 0.7 (5.8 ( 1.6) 1.3 ( 0.2 29.0 ( 2.5 10.0 ( 0.5 40.4 ( 3.8

b

a Spectral Kd values reported for 16–19 are averages obtained from triplicate analysis. b Previously reported value given in parentheses.25

Next, compounds 16–19 were examined as inhibitors for fulllength iNOS. NOS activity was measured using a spectral assay making use of the NO-induced oxidation of oxymyoglobin to metmyoglobin observable at 405 nm. Table 3 lists the Ki values determined for compounds 16–19 (with graphic depictions of the iNOS inhibition assays for all four analogs provided in Supporting Information). Table 3. Ki Values Determined for 16–19 compound G

b

N -amino-L-arginine (16) NG-methylamino-L-arginine (17) NG-amino-NG-methyl-L-arginine (18) NG-amino-NG-2,2,2-trifluoroethyl-L-arginine (19)

Kia (µM) 2.2 ( 0.5 (1.7 ( 0.6) 16.4 ( 5.0 34.1 ( 7.2 10.5 ( 5.7

a Values reported are averages obtained from triplicate analysis. b Previously reported value given in parentheses.26

NOS binding affinities for the four NG-amino-L-arginine analogues were determined using a previously described spectral binding assay.25 For this analysis, a truncated construct of iNOS (iNOSheme, comprised of amino acids 1–490) was used. This construct has been previously shown to bind heme, H4B, and

Compounds 16–19 were next investigated as time-dependent inactivators of iNOS. Each compound was used at a concentration 10-fold higher than the measured Ki and allowed to react with iNOS under turnover conditions (NADPH, H4B, O2). Time points were obtained by quenching aliquots from each reaction mixture into a concentrated L-arginine solution (final concentration of L-arginine 100-fold higher than compound tested). The oxymyoglobin assay was then used to measure residual iNOS activity as a function of preincubation time with 16–19. Of the four compounds tested, only NG-amino-L-arginine 16 (as expected21) and NG-amino-NG-methyl-L-arginine 18 displayed an ability to irreversibly inactivate iNOS in a time-dependent manner. Kinactivation values were determined by plotting residual iNOS activity as a function of preincubation time (Figure 1). The Kinactivation value measured for NG-amino-L-arginine 16 was 0.18 ( 0.01 min-1 (compared to a previously reported value21 of 0.26 min-1). Compound 18 inactivates iNOS at a higher rate with a measured Kinactivation value of 0.39 ( 0.02 min-1. As mentioned above, Osawa and co-workers have shown that NG-amino-L-arginine inactivates NOS by covalent modification of the heme cofactor.22 To investigate whether or not a heme

NG-Aminoguanidines from Primary Amines

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Figure 1. Time-dependent inactivation of iNOS by 18.

modification mechanism was responsible for the iNOS inactivation observed with 18, a mass spectral analysis was used. iNOS was reacted with either NG-amino-L-arginine or compound 18 in the presence of NADPH for 30 min after which the heme content of the samples was assessed by LC-MS. While treatment of iNOS with NG-amino-L-arginine led to formation of the expected adduct22 (MW ) 775.3), no modified heme species were detected in iNOS samples treated with 18. This suggests that heme modification is not an inactivation pathway common to all NG-aminoguanidine-based inhibitors of NOS. The irreversible iNOS inactivation caused by compound 18 is possibly due to modification of an amino acid(s) in the enzyme active site. Compounds 17 and 19 were shown not to be time-dependent inactivators of iNOS and were further examined for an ability to inhibit iNOS-mediated NADPH oxidation (Figure 2). The rate at which NADPH is oxidized by NOS is regulated by a number of factors27 and in the absence of L-arginine the enzyme consumes NADPH (Figure 2, basal activity) to generate reactive oxygen species like superoxide and peroxide.27,28 While incubation with compound 17 led to no significant difference versus the basal rate (no L-arginine present) of iNOS-mediated NADPH consumption, compound 19 severely inhibited NADPH oxidase activity.

Figure 2. Effect of 17 and 19 on iNOS-mediated NADPH consumption. Compound 17 has little impact on the rate of NADPH consumption by iNOS (similar to basal oxidation rate). Compound 19 causes a significant decrease in the observed rate of NADPH oxidation (1 h to equilibrate the reconstituted protein in an O2-free environment. An aliquot of the protein (200 µL) was then reduced with sodium dithionite (50 µM) and mixed with either L-arginine or compound 19 (10 mM) and 100 mM HEPES (pH 7.5) to give a final volume of 1.0 mL. The protein was rapidly mixed with an equal volume of a solution of O2-saturated buffer (2.2 mM). The system had been previously scrubbed of oxygen with a solution of 500 µM sodium dithionite in 100 mM HEPES (pH 7.5). After mixing, the sample was monitored spectrally from 350–650 nm. Final solution concentrations consisted of iNOSheme (2–4 µM), H2B (50–100 µM), DTT (50–125 µM), L-arginine or compound 19 (5 mM), and O2 (1.1 mM). All data was fit with Specfit global analysis software from Spectrum Software Associates (Chapel Hill, NC, USA).

Acknowledgment. This work was supported by the Aldo DeBenedictis Fund, the Natural Sciences and Engineering Council of Canada, and the Alberta Heritage Foundation for Medical Research (postdoctoral fellowships to N.I.M.). Nathaniel Fernhoff and Michelle Chang are gratefully acknowledged for providing full-length iNOS. Supporting Information Available: 1H and 13C NMR spectra for compounds 16–19 and complete results of the spectral binding and inhibition assays for compounds 16–19. This material is available free of charge via the Internet at http://pubs.acs.org.

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